Drought, cold, and salinity are critical environmental factors that limit crop productivity worldwide. In response to these stress signals, plants mount a number of defense reactions that increase the tolerance to the stressful conditions. The best-known molecular response is the activation of gene expression that leads to changes in protein profiles and physiological processes. The products of these so called “stress genes” are categorized in at least two functional groups. The first group includes metabolic enzymes required for biosynthesis of various osmo-protectants, chaperons, detoxifying enzymes, and other effector proteins that are directly involved in stress protection processes. The second category of gene products includes sensor/receptor proteins, protein kinases and phosphatases, enzymes involved in second messenger metabolism, and other components that constitute the signal transduction pathways connecting the stress signal to the cellular responses (Shinozaki et al., Plant Physiol. 115:327-334 (1997); Ingram et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 47:377-403 (1996); Bray, Trends Plant Sci. 2:48-54 (1997)).
In the stress signaling pathways, a number of studies implicate Ca2+ as second messenger. First, elevation of cellular Ca2+ is a rapid response to osmotic signals such as cold, drought, and salinity (Knight et al. Plant J. 12:1067-1078 (1997); Knight et al. Plant Cell 8:489-503 (1996); Knight et al. Nature 352:524-526 (1991)). Secondly, Ca2+ is required for stress-induced gene expression in plants (Knight et al. Plant Cell 8:489-503 (1996)). Thirdly, Ca2+ elevation is sufficient for activation of stress gene expression (Sheen, Science 274:1900-1902 (1996)). It is generally believed that Ca2+ transmits the stress signal downstream in the pathway by interacting with protein sensors. These calcium sensors such as calmodulin (CaM), Ca2+-dependent protein kinases (CDPK), bind Ca2+ and interact with and regulate the activity of their target proteins (Sander et al Plant Cell 11:691-706 (1996); Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414 (1992); Zielinski, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:697-725 (1998)).
Ca2+ serves as a second messenger in many stress signal transduction pathways (Sander et al. Plant Cell 11:691-706 (1996); Trewavas, Plant Physiol. 120:1-6 (1999); Bush, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:95-122 (1995)). However, the molecular mechanism for Ca2+ action in plant cells is not well understood. The present invention demonstrates that a CBL calcium sensor is highly stress-responsive at the protein level. Constitutive expression of a CBL protein is sufficient for activation of multiple stress response pathways leading to enhanced tolerance to both salinity and osmotic stress in transgenic plants. This finding suggests that CBL proteins are an upstream regulator of stress gene expression and serves as a rate-limiting factor in stress response pathways.
One of the earliest responses to stress signals is elevation in cellular calcium in plants (Knight et al. Plant J. 12:1067-1078 (1997); Bush, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:95-122 (1995)). Later responses include changes in expression profiles of a large number of stress genes (Shinozaki and Yamaguchi-Shinozaki, Plant Physiol. 115:327-334 (1997); Ingram et al. Ann Rev. Plant Physiol. Plant Mol. Biol. 47:377-403 (1996); Bray, Trends Plant Sci. 2:48-54 (1997))). From calcium to gene expression, a number of intermediate components may play a role in the signaling process. These include calcium sensors such as CaM, CDPK, and AtCBL that are immediate downstream components following calcium changes (see, Roberts and Harmon Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414 (1992); Zielinski Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:697-725 (1998); and Kudla et al. Proc. Natl. Acad. Sci. USA 96:4718-4723 (1999)).
It is generally believed that cellular Ca2+ levels hold the key to the activation of stress response pathways. Normally, cellular Ca2+ fluctuates below the “threshold value”. Stress signals typically boost the Ca2+ levels over the threshold leading to the activation of calcium sensors and the downstream components in the pathway. Consistent with this view, elevation of cellular Ca2+ by ionophores is sufficient for activation of stress- and ABA-induced gene expression in the absence of stress or ABA application (Sheen, Science 274:1900-1902 (1996)). Levels of calcium sensors may alter the sensitivity to cellular Ca2+. In the case of AtCBL1, its basal level is quite low and activation of the pathway increases the protein level. The stress-responsive increase may lower the threshold value for cellular Ca2+ and serve as a feedback mechanism for increasing the sensitivity to stress signals. In transgenic plants, AtCBL1 protein levels are constantly high so that stress response pathways are turned on by basal Ca2+ levels that are present under “normal” conditions. As a result, these plants are “hypersensitive” to stress signals.
A family of novel calcium sensors from Arabidopsis was recently described (Kudla et al. Proc. Natl. Acad. Sci. USA 96:4718-4723 (1999)). These proteins are similar to both the regulatory B subunit of calcineurin and the neuronal Ca2+ sensor (NCS) in animals (Olafsson et al. Proc. Natl. Acad. Sci USA 92:8001-8005 (1995); Klee et al. J Biol. Chem. 273:13367-13370 (1998)). These unique plant Ca2+ sensors have been referred to as Arabidopsis calcineurin B-like (AtCBL) proteins (Kudla et al. Proc. Natl. Acad. Sci. USA 96:4718-4723 (1999); Shi et al. Plant Cell 11:2393-2405 (1999); Kim et al. Plant Physiol. 124:1844-1853 (2000)). One member of the AtCBL gene family, AtCBL1, is highly inducible by stress signals including drought, cold, and wounding (Kudla et al. Proc. Natl. Acad. Sci. USA 96:4718-4723 (1999)), implicating AtCBL1 in plant response to multiple stress conditions. Another member, AtCBL4 or salt-overly sensitive 3 (SOS3) has been shown to play a role in salt resistance in Arabidopsis (Liu and Zhu, Science 280:1943-1954 (1998)). Like calmodulin (CaM), calcineurin B, and NCS, the AtCBL family calcium sensors are small Ca2+-binding proteins that do not have enzymatic activity by themselves and function as Ca2+-sensors by interacting with other signaling proteins. Using yeast two-hybrid screening, Shi et al. Plant Cell 11:2393-2405 (1999) identified a group of novel protein kinases (CIPKs) as target proteins for AtCBL1. Halfter et al. Proc. Natl. Acad. Sci. USA 97:3735-3740 (2000) describes a similar group of protein kinases (SIP) as targets for AtCBL4/SOS3. CIPK/SIP represents a new subclass of protein kinases that are related to SNF1/AMPK family in the kinase domain but contain a unique regulatory domain in the C-terminal non-kinase region. The C-terminal regulatory domain is required and sufficient for interaction with AtCBL Ca2+ sensors (Shi et al. Plant Cell 11:2393-2405 (1999); Kim et al. Plant Physiol. 124:1844-1853 (2000); Halfter et al. Proc. Natl. Acad. Sci. USA 97:3735-3740 (2000)). Taken together, these studies suggest that the AtCBL family of calcium sensors functions by interacting with a family of protein kinases. The specificity of interaction between the AtCBL calcium sensors and their target kinases is important in determining their specific function in plants (Kim et al. Plant Physiol. 124:1844-1853 (2000)).
To date, only a few studies have been reported on the function of these calcium sensors in stress-induced gene expression. One study shows that constitutive activation of specific CDPK isoforms is sufficient for expression of ABA-induced genes without ABA treatment (see, Sheen Science 274:1900-1902 (1996)). Overexpression of two pathogen-inducible CaM genes activates pathogen-response pathway in a pathogen-independent manner (see, Heo et al. Proc. Natl. Acad. Sci. USA 96:766-771 (1999)). These studies suggest that CaM and CDPK are upstream of pathogen- and ABA-induced gene expression in the signaling pathways.
In light of the above, it is thought that CBL genes are involved in controlling a plant stress response. It is not known, however, whether control of the expression of the genes would be useful in controlling a plant stress response. The present invention addresses these and other needs in the prior art.